Anti-inflammatory properties of butyrate-producing atypical Escherichia coli in a murine colitis model

Ji Hyung Kim; Jee In Yoo; Hyun Woo Ma; I Seul Park; Mijeong Son; Yoojin Shin; Ki Beom Kim; Seung Won Kim; Si Jae Park; Jihye Park

doi:10.5217/ir.2022.00112

Inflammatory bowel disease (IBD) is a chronic refractory intestinal inflammatory disorder and its pathogenesis is related with several factors including genetic, environmental, microbiota, and immune response, but remains largely unknown. In recent decades, gut microbiota composition and function have become a relevant aspect of IBD pathogenesis and expected promising therapeutic options including probiotics and fecal microbiota transplantation [1-3]. Yoon et al. [4] found a novel commensal Escherichia coli strain named atypical E. coli (atEc), which demonstrated anti-inflammatory effect because of 1 additional catalase gene compared to typical E. coli [3,4]. Also, the short-chain fatty acids produced by gut microbiota, such as acetate, propionate, and butyrate are expected to maintain intestinal homeostasis [5,6]. We hypothesized to design a novel butyrate-producing recombinant atEc strain using a 2-plasmid system and demonstrate its anti-inflammatory effect in murine colitis model.

A novel recombinant butyrate atEc strain was designed using a 2-plasmid system capable of introducing foreign genes and overexpressing genes for gene expression in the butyric acid metabolic cycle (Supplementary Fig. 1, Supplementary Table 1).[7]. Animals were randomly divided into 4 groups; control+phosphate buffered saline (PBS) (n = 3), dextran sulfate sodium (DSS)+PBS (n = 4), DSS+atEc (n = 4), and DSS+butyrate atEc groups (n = 4). Male C57BL/6 mice (8-week-old) were treated with 1 × 10⁸ bacteria in 0.2 mL PBS daily by oral gavage before and after the induction of DSS colitis (from day 0 to day 12) (Fig. 1A). DSS (2.5% [w/v]; MP Biomedicals, Solon, OH, USA) was added to their drinking water from day 3 of gavage and continued for day 9, and water was changed to DSS-free water before euthanization. All mice were sacrificed 12 days after the onset of the study. Mice were monitored for survival, body weight loss, disease activity index, and colon length. Parts of the distal colon were cut into 2 to 3 pieces for periodic acid-Schiff staining and histologic scoring was obtained. All animal testing complied with all applicable Korean laws and was approved by the Institutional Animal Care and Use Committee of Yonsei University Severance Hospital (Seoul, Korea) (IACUC No. 2019-0241). The experiments complied with the approved IACUC guidelines. All results are expressed as standard error of the mean. GraphPad Software (La Jolla, CA, USA) was used for all analyses. The significance of differences between conditions was assessed using one-way or two-way analysis of variance followed by Holm-Sidak or Tukey’s multiple comparison test, respectively. P-values < 0.05 were considered significant.

DSS+atEc group attenuated body weight loss (Fig. 1B), disease activity index (Fig. 1C), and histopathologic score (Fig. 1E) compared to DSS+PBS group. However, DSS+butyrate atEc group failed to ameliorate DSS colitis compared to DSS+PBS group (Fig. 1B-F). Also, only 1 mouse died after the 10th day in the DSS+butyrate atEc group during the experiment (Fig. G).

Butyrate-producing bacteria have been expected to have a therapeutic potential in IBD patients to promote intestinal barrier function and modulate immune responses [8]. Previously, we proved the anti-inflammatory effect of atEc in murine colitis model [2]. We also developed a novel recombinant butyrate atEc strain using 2 plasmid system and demonstrated its butyrate-producing ability at the cellular level. However, we failed to demonstrate the anti-inflammatory effect of butyrate atEc in vivo. First, contrary to our hypothesis, butyrate may not only have a good effect, but also have a bad effect on the gut. Zhang et al. [9] found that colonic short-chain fatty acid in certain chemical microenvironment could activate the growth and motility of pathosymbionts E. coli strain. Second, although butyrate was well produced by butyrate atEc strain in vitro setting, there is a possibility that it was not well produced in the intestinal environment affected by various confounders. Measurement of luminal butyrate level could be helpful to demonstrate the effect of butyrate at E. coli strain in vivo. Further study with butyrate-producing atEc strain with the pretreatment of antibiotics may increase the stable colonization of this bacteria overcoming the normal flora present. Third, a genetically recombined butyrate-producing strain can affect the composition of various metabolites and microbiomes in the gut, which can affect outcomes. Butyrate production efforts with metabolically engineered E. coli under oxygen-limited conditions had limited success in previous studies [10]. Therefore, further studies should be warranted to develop a novel strain that works in the intestinal environment.

Notes

Funding Source

This research was supported by Young Medical Scientist Research Grant through the Daewoong Foundation (2021-31-1199) and National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1A2C 2003638 and NRF-2020R1F1A1070249).

Conflict of Interest

Park J is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.

Data Availability Statement

All study-related data is included in the publication or provided as supplementary information.

Author Contributions

Conceptualization: Park J, Park SJ. Data curation: Kim JH, Park J. Formal analysis: Kim JH. Funding acquisition: Park J, Park SJ. Investigation: Kim JH, Yoo JI, Park J, Ma HW, Park IS, Son M, Shin Y, Kim KB. Methodology: Kim JH, Yoo JI, Kim SW. Project administration: Park J. Resources: Kim SW. Software: Kim JH, Park J. Supervision: Park J, Park SJ. Validation: Kim SW. Visualization: Kim JH. Writing – original draft: Park J. Writing – review & editing: Kim JH, Kim SW. Approval of final manuscript: all authors.

Supplementary Material

Supplementary materials are available at the Intestinal Research website (https://www.irjournal.org).

Supplementary Fig. 1. Two plasmid systems used for the production of butyrate. (A) Structure of constructed plasmid vectors and preparation of butyrate-producing recombinant atEc strain. Plasmids pKM212-ter-crt-tesB and pKA312-hbd-atoB were used for the production of butyrate and transformed into atEc strain. Bacterial strains and plasmids used in this study were described in Supplementary Table 1. All bacterial strains used for the mice group were incubated with appropriate antibiotics and pre-cultured in 25 mL test tubes containing 10 mL LB medium at 37ºC, 16 hours in a rotary shaker at 220 rpm. One milliliter of 16 hours culture was used to inoculate 250 mL flask containing 100 mL LB medium, recombinant microbial strains were induced by 0.05 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the absorbance (OD600) reached about 0.4. Afterward, induced culture cells were harvested at the absorbance (OD600) reached about 1, and 1 mL of each culture medium was washed twice and suspended with 1 mL PBS solution. (B) Butyrate production in recombinant atEc strains. Butyrate productions from bacteria were measured using HPLC (Agilent, Palo Alto, CA, USA) as the previous report. [7] Butyrate-atEc strain was used to DSS-induced colitis mouse model. atEc, atypical Escherichia coli-treated; DSS, dextran sulfate sodium DSS-treated.

ir-2022-00112-suppl1.pdf

Supplementary Table 1. Bacterial Strains and Plasmids Used in This Study

ir-2022-00112-suppl2.pdf

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Fig. 1.

Anti-inflammatory effects of butyrate-producing atypical Escherichia coli in murine DSS colitis model. (A) Scheme of the experiment. Eight-week-old male C57BL/6 mice were treated 1×10⁸ bacteria or PBS daily by oral gavage. (B) Body weight was checked during the 12 days before and after induction of colitis by 2.5% DSS added to the drinking water from day 3 of gavage and continued for 7 days. All mice were euthanized on day 12 of the study and disease activity index (C), colon length (D), periodic acid-Schiff staining (E), histopathological score (F), and survival rates (G) were analyzed. Data are expressed as the mean±standard error of the mean (n=4). Analyses were performed using two-way analysis of variance (ANOVA) followed by Tukey multiple comparison test (B, C) and one-way ANOVA followed by Holm-Sidak multiple comparison test (D, F). ^avs. control: P<0.001, ^b-dvs. DSS+PBS group or between indicated groups: ^bP<0.05, ^cP<0.01, ^cP<0.001. PBS, phosphate buffered saline-treated vehicle; DSS, dextran sulfate sodium treated; atEc, atypical E. coli-treated; butyrate atEc, butyrate atypical E. coli-treated.